We acknowledge the technical assistance of the Translational Research Institute Histology core and Biological Resource Facility. This research was supported by funding from a Princess Alexandra Research Foundation award (I.V., E.D.W.), and the Medical Research Future Fund (MRFF) Rapid Applied Research Translation Program (Centre for Personalised Analysis of Cancers (CPAC; E.D.W., I.V.). The Translational Research Institute receives support from the Australian Government.

Patients have consented to this study following their admission under the Urology team at the Princess Alexandra Hospital, Brisbane, Australia. This study was performed in accordance with the principles of the Declaration of Helsinki and within ethical and institutional guidelines (ethics number HREC/05/QPAH/95, QUT 1000001165).
NOTE: As eligibility criteria, patients were aged &#8805; 18 with cancer, and able to understand and provide consent. Those who were not able to give informed consent were excluded. Those having a primary language other than English were excluded as the provision of interpreters was not possible due to logistical and budgetary considerations. Also excluded were patients whose tumors were not accessible to biopsy or unlikely to be available in adequate amount after routine pathology.
1. Organoid Medium Preparation
NOTE: Human bladder cancer organoid medium requires growth factors that aid in the survival, growth, and continuous expansion of organoids derived from dissociated clinical material (Table 1). For complete details of each supplement used in this procedure, please refer to the Table of Materials.
<ol>
	<li>Thaw frozen ingredients on ice or in a refrigerator at 2-8 &#176;C. Avoid repeated freeze/thaw cycles and work from frozen aliquots (stored at -20 &#176;C).</li>
	<li>Basal medium: Prepare basal medium by supplementing advanced Dulbecco&#39;s Modified Eagle Medium/Ham&#39;s F-12 (adDMEM/F12) with HEPES (10 mM) and glutamine (2 mM). This is also used as a transport medium, and during organoid washing steps. 
	NOTE: Advanced DMEM/F-12 is used as the basal medium to aid in the expansion of organoids in the presence of limited serum; however, it requires supplementation with HEPES and L-glutamine.</li>
	<li>Briefly centrifuge fibroblast growth factor 10 (FGF-10), fibroblast growth factor 2 (FGF-2), epithelial growth factor (EGF), SB202190, prostaglandin E2 (PGE2), and A 83-01 before opening to ensure components are at the bottom of the vial.</li>
	<li>Complete organoid medium: Supplement basal medium with human noggin- and R-spondin 1-conditioned media at a final concentration of 5% v/v, human EGF (50 ng/mL), human FGF-2 (5 ng/mL), human FGF-10 (20 ng/mL), A 83-01 (500 nM), SB202190 (10 &#181;M), B27 (1x), nicotinamide (10 mM), N-acetylcysteine (1.25 mM), Y-27632 (10 &#181;M), PGE2 (1 &#181;M) and a broad-spectrum antibiotic formation at the concentration indicated by the manufacturer.</li>
	<li>Store organoid media at 4 &#176;C in the dark and use it within 2 weeks (1 month at most). Do not freeze. Avoid extended exposure to light sources. 
	NOTE: The organoid medium is prepared serum-free; however, serum and penicillin/streptomycin could be supplemented on a user-to-user basis as required.</li>
</ol>
2. A day before procedure outlined in 3
<ol>
	<li>Thaw growth factor reduced basement membrane (BME; see Table of Materials) overnight for at least 12 h before use in a 4 &#176;C refrigerator or cold room. If required, dispense BME into 1 mL aliquots in a 1.5 mL polypropylene single-use tube to avoid freeze-thaw cycles.</li>
	<li>Place filtered pipette tips into a 4 &#176;C refrigerator or cold room. 
	NOTE: This section refers to pipette tips that will be used when handling BME to prevent premature polymerization and reduce the coating of BME on the surface of the tips.</li>
	<li>Sterilize all surgical equipment required for the procedure.</li>
</ol>
3. Generation of bladder tumor organoids
NOTE: This is an initial step for PDO establishment from primary patient tumors. This procedure is adapted for bladder cancer tissues from methods established by Gao et al.24.
<ol>
	<li>Use a class II biohazard hood to prepare the specimen. Retrieve dry and wet ice and print Patient Specimen Processing Sheet (Supplemental File).</li>
	<li>Call research personnel to the operating room when surgical resection is near complete. 
	NOTE: Confirm that the patient meets the eligibility criteria and has signed the participant consent form. Tissue is provided for research only if surplus to requirements for clinical histopathologic assessment.</li>
	<li>Collect a fresh macroscopically viable tumor specimen from surgery. Ensure that the sample is submerged in transport medium (either 1x adDMEM/F12 or 1x Dulbecco&#39;s phosphate-buffered saline (DPBS)) in a sterile 50 mL conical tube or urine specimen jar during transit. 
	NOTE: At some clinical centers, tissue may need to be transported to a pathology laboratory for to be allocated for research. Under these circumstances, it is recommended that antibiotics and antimycotics be added to the transport medium. Tissue can be stored at 4 &#176;C in basal medium for up to 24 h post-surgery and still generate viable organoid cultures.</li>
	<li>Record specimen details, including tissue weight (g or mg), sample description, and details regarding any blood and urine samples on the Patient Specimen Processing Sheet (Supplemental File).</li>
	<li>Carefully remove the transport medium and replace it with 10 mL of basal media. Allow the tumor tissue to settle by gravity. 
	NOTE: Transport medium is considered as clinical waste and must be collected in an appropriately labeled waste container in an appropriate amount of decontamination solution. Once the liquid has been chemically decontaminated, it can be disposed of according to institutional guidelines for hazardous waste.</li>
	<li>Remove the tumor tissue with forceps and place it in a sterile 90 mm Petri dish (Figure 2A). Record the weight of the tissue in mg or g on the clinical specimen processing sheet and dissection grid (Figure 2B).</li>
	<li>Remove non-cancerous tissue (including adipose tissue) and macroscopically visible necrotic regions using sterile forceps and disposable scalpel blade mounted to scalpel handle (Figure 2C). Wash tumor pieces 1-2 times with cold 1x DPBS. Collect the tumor pieces and transfer them to a new sterile 90 mm Petri Dish. 
	NOTE: As scalpel blades are sharp, take caution when manually slicing. Tumor-adjacent adipose tissue can be identified by distinctly soft, gelatinous, pale areas immediately adjacent to the visual tumor perimeter. Macroscopic adipose tissue, and focally dark regions representing areas of necrosis will require personal judgment when dissecting from an excisional bladder tumor biopsy.</li>
	<li>Take a photograph, draw a diagram of tissue, and plan tissue dissection on a clinical processing grid (Figure 2B and Figure 2C). 
	NOTE: It is important to keep a visual record and rough sketch of the individual macroscopic tumor characteristics and dissection for each specific case.</li>
	<li>Dissect tumor tissue pieces and allocate for histopathological (Figure 2D) and molecular analyses (Figure 2E).
	<ol>
		<li>For histological analyses: Place approximately 50 mg of tumor tissue into a labeled disposable plastic histology cassette (Figure 2D). Submerge histology cassette into a container with 5x to 10x volume of 10% neutral buffered formalin (NBF). Incubate overnight at RT.</li>
		<li>Remove 10% NBF and replace with 70% (w/w) ethanol the next day for storage at 4 &#176;C until tissue can be processed using routine tissue processing protocol</li>
		<li>For molecular analyses: Snap freeze at least one 1-3 mm3 tumor piece in an RNase/DNase-free 1.5 mL cryovial using liquid nitrogen and store at -80 &#176;C (Figure 2E).</li>
	</ol>
	</li>
	<li>Dispense 5 mL of organoid medium (Table 1) in the 90 mm Petri dish containing the remaining tumor piece(s).</li>
	<li>Mechanically mince tissue as finely as possible (0.5-1 mm3 pieces or smaller) with a sterile #10 scalpel blade. 
	NOTE: Larger fragments or whole chunks of tissue (&#62;3 mm3) will take considerably longer to digest and will decrease the viability of the specimen. Omit the above step if the tissue is disaggregated and fragments are small enough to pipette with a 5 mL serological pipette tip.</li>
	<li>Transfer finely minced tissue to a 50 mL conical tube and add 4 mL of organoid medium, 1 mL of 10x collagenase/hyaluronidase, and 0.1 mg/mL deoxyribonuclease 1 (DNase 1) to avoid cell clumping. 
	NOTE: Enzyme solution should be freshly prepared each time. Increase the volume of the medium to be approximately 10x the visible amount of the tumor fragments for large samples.</li>
	<li>Incubate the minced tumor tissue and enzyme solution for 1-2 h on an orbital shaker or rotator (150 rpm) in an incubator (37 &#176;C, 5% CO2) to dissociate fragments into a cell suspension and break down collagens. This can be checked with histological analysis (Figure 3A). 
	NOTE: If the amount of tissue is large or no obvious dissociation is observed after 1-1.5 h, increase the incubation time checking the level of dissociation every 30 min. Timing for this step depends on the sample and must be determined empirically each time. A noticeably clearer solution with tissue fragments indiscernible to the eye (or very few fragments) indicates successful digestion.</li>
	<li>Terminate the digestion with the addition of 2x volume (20 mL) of basal medium to the sample.</li>
	<li>Centrifuge the sample at 261 x g for 5 min at room temperature (RT), aspirate, and discard the supernatant.</li>
	<li>To lyse contaminating red blood cells (RBCs), resuspend the pellet obtained from the above step in 5 mL of ammonium-chloride-potassium (ACK) buffer. Incubate the tube at RT for 3 min or until the complete lysis of the RBCs is seen (suspension becomes clear). 
	NOTE: If RBCs are not observed as a small red clump in the pellet, this step may be omitted.</li>
	<li>Add 20 mL of the basal medium into the tube. Centrifuge the tube at 261 x g for 5 min at RT and aspirate the supernatant.</li>
	<li>At this step, place a 10 mL aliquot of 2x and 1x organoid medium in a 37 &#176;C water bath to warm.</li>
	<li>Filter the sample through a pre-wet reversible 100 &#181;m strainer into a new 50 mL tube to remove large insoluble material. 
	NOTE: Large undigested material (&#62;100 &#181;m) collected by the filter may contain cells of interest and can be cultured in 90 mm Petri dish or 6-well cell culture plate in organoid medium to derive 2D cultures (Figure 1 (step 5) and Figure 3B).</li>
	<li>Filter the eluate through a pre-wet reversible 37 &#181;m strainer to collect single cells and small clusters for single-cell and immune cell isolation (Tube 37-1; Figure 1 (step 6) and Figure 3B). 
	NOTE: The yield of tumor cells during this step may be increased by passing the filtered cell suspension through the strainer again.</li>
	<li>Reverse the 37 &#181;m strainer and use 10 mL of basal media to collect small and moderate-sized clusters (37-100 &#181;m) (tube 70-1; Figure 3B).</li>
	<li>Top up each of the new 50 mL tubes (tubes 70-1 and 37-1) with DPBS to 40 mL. Centrifuge the suspension at 261 x g for 5 min at RT. Aspirate and discard the supernatant.</li>
	<li>Add 10 mL of basal media to tube 37-1 and count the cells using trypan blue exclusion dye and an automated cell counter (as per the manufacturer&#39;s specifications). Determine the cell number and viability of cells.</li>
	<li>Centrifuge the remainder of the sample at 261 x g for 5 min at RT. Aspirate the medium and replace it with cell freezing solution or basal media containing 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, and 10% dimethyl sulfoxide (DMSO).</li>
	<li>Place samples in 1.5 mL cryovials and store them in a cell-freezing container. Immediately transfer the container to a -80 &#176;C freezer for optimal rate of cooling. Following overnight freezer storage at -80 &#176;C, transfer the cryovials into cryogenic liquid or air phase storage (-196 &#176;C) for long-term storage.</li>
	<li>Resuspend cells from tube 70-1 with 500 &#181;L of pre-warmed 2x organoid medium. 
	NOTE: Seeding density must be high for successful organoid propagation. The volume of the organoid medium and, subsequently, BME should be altered empirically on the number of cells isolated from the filtration step. This step provides a relevant starting point based on studies in our laboratory.</li>
	<li>Add BME to cells (at a 1:1 ratio with 2x organoid medium) with ice-cold P1000 sterile filter pipette tips and mix gently to suspend cells. Quickly and carefully pipette 100 &#181;L of reconstituted cells/ BME mixture to wells of an ultra-low attachment flat-bottom 96-well plate. Place the 96-well plate in an incubator (37 &#176;C, 5% CO2) for 20-30 min to solidify.</li>
	<li>Add 1:2 ratio of 1x organoid medium on top of BME cell suspension depending on the empirically assessed volume. In the relevant starting point, add 50 &#181;L of 1x organoid medium media on top of 100 &#181;L of reconstituted cells/ BME mixture.</li>
	<li>Place the 96-well plate in an incubator (37 &#176;C, 5% CO2) for 20 min to equilibrate.</li>
	<li>Take the cell culture plate out of the incubator and assemble it on a specimen holder on the stage of a microscope. Assess organoids visually under phase contrast or brightfield settings. 
	NOTE: Images are best acquired by an inverted phase-contrast microscope equipped with differential interference (DIC) optics, digital camera, and associated software to observe the formation of spherical PDOs in preparation for endpoint analysis.
	<ol>
		<li>If distinct clusters are visible, place the cell culture plate into an onstage electronically heated microscope chamber (37 &#176;C, 5% CO2) for live-cell imaging over the first 24-72 h.</li>
		<li>Ensure the flexible heated collar is attached to the lens to reduce thermal drift and the humidifier is filled with dH2O.</li>
		<li>Perform the initial imaging using a 4x or 10x objective lens (N.A. 0.30, W.D. 15.2 mm). Time-lapse every 5-10 min on the phase-contrast setting.</li>
		<li>Check for the successful isolation as characterized by the appearance of &#62;10 self-organizing organoids per well after a 24-72 h period.</li>
		<li>Allow cultures to continue for up to two weeks if organoid numbers are low (Figure 3C).</li>
	</ol>
	</li>
	<li>Top up the medium every 2-3 days using 50 &#181;L of pre-warmed organoid medium to replenish depleted growth factors and overall volume.</li>
	<li>Acquire images (as described in step 3.30) on days 1, 2, and 3 (time-lapse series), and on days 5, 7, and 10 before passaging (if applicable). 
	NOTE: Organoids (observed as generally round structures where you cannot see the edges of individual cells) are typically passaged 7-10 days following setup, depending on the success of the isolation. Before or following passaging, organoids can be treated with cytotoxics or therapeutic agents for up to 6 days and drug response measured using cell viability and cytotoxicity assays. Alternatively, organoids can be retrieved from BME (using 1 mg/mL dispase) and cryopreserved (bio banked), prepared for molecular testing, or embedded and assessed histologically (Figure 4).</li>
</ol>

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(155), e60364 (2020).</li><li>Pleguezuelos-Manzano, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+culture+of+human+intestinal+organoids+derived+from+adult+stem+cells.">Establishment and culture of human intestinal organoids derived from adult stem cells.</a> Current Protocols in Immunology. 130 (1), 106 (2020).</li><li>Fusco, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-derived+organoids+(PDOs)+as+a+novel+in+vitro+model+for+neuroblastoma+tumours.">Patient-derived organoids (PDOs) as a novel in vitro model for neuroblastoma tumours.</a> BMC Cancer. 19 (1), 970 (2019).</li><li>Mullenders, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+and+human+urothelial+cancer+organoids:+A+tool+for+bladder+cancer+research.">Mouse and human urothelial cancer organoids: A tool for bladder cancer research.</a> Proceedings of the National Academy of Sciences. 116 (10), 4567-4574 (2019).</li><li>Vasyutinv, I., Zerihun, L., Ivan, C., Atala, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bladder+organoids+and+spheroids:+potential+tools+for+normal+and+diseased+tissue+modelling.">Bladder organoids and spheroids: potential tools for normal and diseased tissue modelling.</a> Anticancer Research. 39 (3), 1105-1118 (2019).</li><li>Santos, C. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Urothelial+organoids+originating+from+Cd49fhigh+mouse+stem+cells+display+Notch-dependent+differentiation+capacity.">Urothelial organoids originating from Cd49fhigh mouse stem cells display Notch-dependent differentiation capacity.</a> Nature Communications. 10 (1), 4407 (2019).</li><li>Whyard, T., Liu, J., Darras, F. S., Waltzer, W. C., Romanov, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoid+model+of+urothelial+cancer:+establishment+and+applications+for+bladder+cancer+research.">Organoid model of urothelial cancer: establishment and applications for bladder cancer research.</a> Biotechniques. 69 (3), 193-199 (2020).</li></ol>

The authors have no conflicts of interest.

While 3D organoid protocols derived from bladder cancer tissue are still in their infancy, they are an area of active research and clinical investigation. Here, an optimized protocol to successfully establish bladder cancer PDOs that are suitable for both NMIBC and MIBC specimens is detailed. This workflow integrates parallelly into hospital-based clinical trials and considers biobank sample accrual, including histological sample processing and fresh frozen tissue banking, which is an important consideration for clinical...

Bladder cancer is the most prevalent urinary tract cancer and the tenth most common human malignancy worldwide1. It encompasses a genetically diverse and phenotypically complex spectrum of disease2. Urothelial non-muscle-invasive forms of bladder cancer (NMIBC) are the most common bladder cancer diagnoses (70%-80%), and these cancers display considerable biological heterogeneity and variable clinical outcomes2,3,4. Patients with NMIBC typically experience a high risk of disease recurrence (50-70%) and one-third of cancers will progress and develop into significantly more aggressive muscle-invasive bladder cancer (MIBC)2. Although 5-year survival rates for NMIBC are high (&#62;90%), these patients must undergo long-term clinical management5. On the other hand, locally advanced (unresectable) or metastatic MIBC is generally considered incurable6. Consequently, bladder cancer has one of the highest lifetime treatment costs within cancer care and is a significant burden for both the individual and the healthcare system3,7. The underlying genetic aberrations in advanced disease renders therapeutic management of bladder cancer a clinical challenge, and therapeutic options for invasive urothelial tumors have only recently improved since the approval of immunotherapies for both advanced and high-risk NMIBC8,9. Currently, clinical decision-making has been guided by conventional clinical and histopathological features, despite individual bladder cancer tumors showing large differences in disease aggressiveness and response to therapy10. There is an urgent need to accelerate research into clinically useful models to improve the prediction of individual patient prognosis and identification of effective treatments.
Three-dimensional (3D) organoids show great potential as tumor models due to their ability to self-organize and recapitulate the original tumor&#39;s intrinsic in vivo architecture and pharmacogenomic profile, and their capability to mirror the native cellular functionality of the original tissue from which they were derived11,12,13. Although established bladder cancer cell lines are readily available, relatively cost-effective, scalable, and simple to manipulate, the in vitro cell lines largely fail to mimic the spectrum of diverse genetic and epigenetic alterations observed in clinical bladder cancers12,14 and were all established and maintained under 2D, adherent culture conditions. Additionally, cell lines derived from primary and metastatic bladder tumors harbor significant genetic divergence from the original tumor material. 8,15.
An alternative approach is to use genetically engineered and carcinogen-induced mouse models. However, while these models recapitulate some of the natural oncogenic cascades involved in human neoplasia (reviewed in refs16,17,18), they lack tumor heterogeneity, are expensive, poorly represent invasive and metastatic bladder cancer, and are not viable for rapid term drug testing as tumors can take many months to develop14,19. Patient-derived models of cancer (including organoids, conditionally reprogrammed primary cell culture, and xenografts) provide invaluable opportunities to understand the effects of drug treatment before clinical treatment20. Despite this, few groups routinely use these patient-proximal models due to limited access to fresh primary patient tissue and the extensive optimization required to reproducibly generate patient-derived organoid (PDO) culture conditions. In an in vivo setting, oncogenic cells can interact and communicate with various compositions of the surrounding constituents, including stromal cells, tissue infiltrating immune cells, and matrix12. Similarly, for PDOs grown in a 3D format, cellular/matrix complexity can be customized to include other relevant components. PDOs can be rapidly generated and are often able to be passaged extensively or cryopreserved for later use, despite having a finite lifespan21,22,23. Pharmacodynamics (i.e., response to a drug) can be evaluated using multiple read-outs, including organoid viability and morphology, and characterization of immunohistochemistry targets or transcriptional changes.
Here, the procedures for the establishment of bladder cancer organoids from patient material collected from transurethral resection of bladder tumor (TURBT) or surgical removal of the bladder (radical cystectomy) are described. The method to generate PDOs is illustrated, using readily available wet laboratory materials and tools. Endpoints include changes in cell morphological characteristics and viability. These were measured using fluorescence microscopy, in vitro viability (metabolic and cell membrane integrity) assays, and histopathological analysis. Figure 1 shows the workflow for establishing human bladder cancer PDOs from clinical material obtained during elective surgery.

<table><tbody><tr><td>1.2 mL cryogenic vial</td><td>Corning</td><td>430487</td><td></td></tr><tr><td>1.5 mL Eppendorf tubes</td><td>Sigma-Aldrich</td><td>T9661-500EA</td><td>polypropylene single-use tube</td></tr><tr><td>100 &amp;micro;M reversible strainer</td><td>STEMCELL Technologies</td><td>#27270</td><td></td></tr><tr><td>100 mm Petri dish</td><td>Corning</td><td>430167</td><td></td></tr><tr><td>10x Collagenase/ Hyaluronidase</td><td>STEMCELL Technologies</td><td>#07912</td><td></td></tr><tr><td>37 &amp;micro;M reversible strainer</td><td>STEMCELL Technologies</td><td>#27250</td><td></td></tr><tr><td>37&amp;deg;C incubator</td><td></td><td></td><td></td></tr><tr><td>37&amp;deg;C water bath</td><td></td><td></td><td></td></tr><tr><td>50 mL falcon tube</td><td>Corning</td><td>CLS430829-500EA</td><td></td></tr><tr><td>6-well plate</td><td>Corning</td><td>CLS3516</td><td></td></tr><tr><td>70% (w/w) ethanol</td><td></td><td></td><td></td></tr><tr><td>-80&amp;deg;C freezer</td><td></td><td></td><td></td></tr><tr><td>96 well ultra low attachment plate (Black)</td><td>Sigma-Aldrich</td><td>CLS3474-24EA</td><td></td></tr><tr><td>A 83-01</td><td>BioScientific</td><td>2939</td><td>Prevents the growth-inhibitory effects of TGF-&amp;beta;</td></tr><tr><td>ACK lysis buffer</td><td>STEMCELL Technologies</td><td>#07850</td><td></td></tr><tr><td>adDMEM/F-12</td><td>Thermo Fisher Scientific</td><td>12634028</td><td>Base medium</td></tr><tr><td>Animal-free recombinant EGF</td><td>Sigma-Aldrich</td><td>518179</td><td>Growth factor</td></tr><tr><td>Automated cell counter (TC20)</td><td>Bio-rad</td><td>1450102</td><td></td></tr><tr><td>B27 additive</td><td>Gibco</td><td>17504044</td><td>Increases sphere-forming efficiency</td></tr><tr><td>Cell Counting Slides</td><td>Bio-rad</td><td>1450015</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>EP022628188</td><td></td></tr><tr><td>Computer system</td><td></td><td></td><td></td></tr><tr><td>CryoStor CS10</td><td>STEMCELL Technologies</td><td>#07930</td><td>Cell freezing solution</td></tr><tr><td>Dispase II, powder</td><td>Thermofisher</td><td>17105041</td><td>To enzymatically disrupt Matrigel</td></tr><tr><td>DNAse 1</td><td>STEMCELL Technologies</td><td>#07900</td><td></td></tr><tr><td>DPBS</td><td>Thermofisher</td><td>14190144</td><td></td></tr><tr><td>Dry and wet ice</td><td></td><td></td><td></td></tr><tr><td>Esky</td><td></td><td></td><td></td></tr><tr><td>Farmdyne (Iodine 16g/L)</td><td>Ecolab</td><td></td><td></td></tr><tr><td>Formalin solution, neutral buffered, 10%</td><td>Sigma</td><td>HT501128</td><td>Histological tissue fixative</td></tr><tr><td>Glutamax (L-alanine-L-glutamine)</td><td>Invitrogen</td><td>35050061</td><td>Source of nitrogen for the synthesis of proteins, nucleic acids</td></tr><tr><td>HEPES</td><td>Gibco</td><td>15630-080</td><td>All-purpose buffer</td></tr><tr><td>Histology cassette</td><td>ProSciTech</td><td>RCH44-W</td><td></td></tr><tr><td>Human FGF-10</td><td>Peprotech</td><td>100-26-25</td><td>Growth factor</td></tr><tr><td>Human FGF-2</td><td>Peprotech</td><td>100-18B-50</td><td>Growth factor</td></tr><tr><td>Liquid nitrogen</td><td></td><td></td><td></td></tr><tr><td>Matrigel (Growth Factor Reduced (GFR), phenol red-free, LDEV free)</td><td>In Vitro Technologies</td><td>356231</td><td>Basement membrane extract (BME)</td></tr><tr><td>Mr. Frosty freezing container</td><td>ThermoFisher</td><td>5100-0001</td><td>cell freezing container</td></tr><tr><td>N-acetyl-L-cysteine (NAC)</td><td>Sigma</td><td>A7250</td><td>Anti-oxidant required to protect against ROS-induced cytotoxicity</td></tr><tr><td>Nicotinamide</td><td>Sigma</td><td>N0636-100G</td><td>SIRT-1 inhibitor</td></tr><tr><td>NikonTs2U inverted microscope</td><td>Nikon</td><td>MFA510BB</td><td></td></tr><tr><td>NIS-Elements Advanced Research</td><td>Nikon</td><td>MQS31000</td><td></td></tr><tr><td>Noggin conditioned media</td><td>In-house</td><td></td><td>BMP inhibitor</td></tr><tr><td>Pipetboy acu 2</td><td>Integra</td><td>155000</td><td></td></tr><tr><td>Pipettes (p20, p100, p1000) with tips</td><td></td><td></td><td></td></tr><tr><td>Primocin</td><td>Jomar Bioscience</td><td>ant-pm2</td><td>Combination of antibacterial and antifungal compounds to protect cell cultures from contaminations</td></tr><tr><td>Prostaglandin E2 (PGE2)</td><td>Tocris</td><td>2296</td><td>support proliferation of cells</td></tr><tr><td>Rotary tube mixer</td><td>Ratek</td><td>RSM7DC</td><td></td></tr><tr><td>R-spondin 1 conditioned media</td><td>In-house</td><td></td><td>WNT signalling regulator</td></tr><tr><td>SB202190</td><td>Jomar Bioscience</td><td>s1077-25mg</td><td>Selective p38 MAP kinase inhibitor</td></tr><tr><td>Scale</td><td></td><td></td><td></td></tr><tr><td>Scalpel handle</td><td>Livingstone</td><td>WBLDHDL03</td><td></td></tr><tr><td>Scalpels, #11 blade</td><td>Medical and Surgical Requisites</td><td>EU-211-1</td><td></td></tr><tr><td>Serological pipettes (5, 10, 25 mL)</td><td></td><td></td><td></td></tr><tr><td>Specimen Waste Bags</td><td>Medical Search</td><td>SU09125X16</td><td></td></tr><tr><td>Urine specimen jar</td><td></td><td></td><td></td></tr><tr><td>Y27632</td><td>Jomar Bioscience</td><td>s1049-10mg</td><td>Selective ROCK inhibitor. Increases survival of dissociated epithelial cells</td></tr></tbody></table>

culture of bladder cancer organoids as precision medicine tools

Current in vitro therapeutic testing platforms lack relevance to tumor pathophysiology, typically employing cancer cell lines established as two-dimensional (2D) cultures on tissue culture plastic. There is a critical need for more representative models of tumor complexity that can accurately predict therapeutic response and sensitivity. The development of three-dimensional (3D) ex vivo culture of patient-derived organoids (PDOs), derived from fresh tumor tissues, aims to address these shortcomings. Organoid cultures can be used as tumor surrogates in parallel to routine clinical management to inform therapeutic decisions by identifying potential effective interventions and indicating therapies that may be futile. Here, this procedure aims to describe strategies and a detailed step-by-step protocol to establish bladder cancer PDOs from fresh, viable clinical tissue. Our well-established, optimized protocols are practical to set up 3D cultures for experiments using limited and diverse starting material directly from patients or patient-derived xenograft (PDX) tumor material. This procedure can also be employed by most laboratories equipped with standard tissue culture equipment. The organoids generated using this protocol can be used as ex vivo surrogates to understand both the molecular mechanisms underpinning urological cancer pathology and to evaluate treatments to inform clinical management.

3D organoids were successfully established from human bladder cancer patient TURBT and cystectomy tissues. Briefly, this technique highlights a rapid formation of 3D multicellular structures that are both viable and suitable for other endpoint analyses such as histological evaluation, molecular characterization (by immunohistochemistry or quantitative real-time PCR), and drug screening. During the procedure (Figure 1), the various eluates during our filtration phases (Fi...

Watch this Scientific Journal Video about Culture of Bladder Cancer Organoids as Precision Medicine Tools at JoVE.com

Culture of Bladder Cancer Organoids as Precision Medicine Tools

Patient-derived organoids (PDOs) are a powerful tool in translational cancer research, reflecting both the genetic and phenotypic heterogeneity of the disease and response to personalized anti-cancer therapies. Here, a consolidated protocol to generate human primary bladder cancer PDOs in preparation for the evaluation of phenotypic analyses and drug responses is detailed.

Current in vitro therapeutic testing platforms lack relevance to tumor pathophysiology, typically employing cancer cell lines established as ...

culture-of-bladder-cancer-organoids-as-precision-medicine-tools

Research

JoVE Journal

Cancer Research

4.6K Views.  Queensland University of Technology (QUT) at Translational Research Institute. Deze methode is belangrijk omdat van patiënten afgeleide organoïden een snel en krachtig hulpmiddel bieden om urologische kankers te bestuderen, omdat ze vaak de genetische en fenotypische heterogeniteit van deze multispectrumziekte nabootsen. Organoïden kunnen worden geïsoleerd uit een beperkte hoeveelheid biopsiemateriaal van de patiënt en kunnen snel worden uitgebreid voor downstream-analyse. De organoïden die met behulp van dit protocol worden gegenereerd, kunnen zowel worden gebrui...

Video: Culture of Bladder Cancer Organoids as Precision Medicine Tools

High-sensitivity Detection of Micrometastases Generated by GFP Lentivirus-transduced Organoids Cultured from a Patient-derived Colon Tumor

Despite current advances in human colorectal cancer (CRC) treatment, few radical therapies are effective for the late stages of CRC. To overcome this clinical challenge, tumor xenograft mouse models using long-established human carcinoma cell lines and many transgenic mouse models with tumors have been developed as preclinical models. They partially mimic the features of human carcinomas, but often fail to recapitulate the key aspects of human malignancies including invasion and metastasis. Thus, alternative models that better represent the malignant progression in human CRC have long been awaited.
We herein show generation of patient-derived tumor xenografts (PDXs) by subcutaneous implantation of small CRC fragments surgically dissected from a patient. The colon PDXs develop and histopathologically resemble the CRC in the patient. However, few spontaneous micrometastases are detectable in conventional cross-sections of affected distant organs in the PDX model. To facilitate the detection of metastatic dissemination into distant organs, we extracted the tumor organoid cells from the colon PDXs in culture and infected them with GFP lentivirus prior to injection into highly immunodeficient NOD/Shi-scid IL2R&#947;null (NOG) mice. Orthotopically injected PDX-derived CRC organoid cells consistently form primary tumors positive for GFP in recipient mice. Moreover, spontaneously developing micrometastatic colonies expressing GFP are notably detected in the lungs of these mice by fluorescence microscopy. Moreover, intrasplenic injection of CRC organoids frequently produces hepatic colonization. Taken together, these findings indicate GFP-labelled PDX-derived CRC organoid cells to be visually detectable during a multistep process termed the invasion-metastasis cascade. The described protocols include the establishment of PDXs of human CRC and 3D culture of the corresponding CRC organoid cells transduced by GFP lentiviral particles.

To allow highly sensitive detection of the disseminating human colorectal cancer (CRC) cells colonizing tissues, we herein show a protocol for efficient transduction of green fluorescent protein (GFP) lentiviral particles into PDX-derived CRC organoid cells prior to their injection into recipient mice, with stereo-fluorescence microscopic observation.

Despite current advances in human colorectal cancer (CRC) treatment, few radical therapies are effective for the late stages of CRC. To overcome this ...

3-D Cell Culture System for Studying Invasion and Evaluating Therapeutics in Bladder Cancer

Bladder cancer is a significant health problem. It is estimated that more than 16,000 people will die this year in the United States from bladder cancer. While 75% of bladder cancers are non-invasive and unlikely to metastasize, about 25% progress to an invasive growth pattern. Up to half of the patients with invasive cancers will develop lethal metastatic relapse. Thus, understanding the mechanism of invasive progression in bladder cancer is crucial to predict patient outcomes and prevent lethal metastases. In this article, we present a three-dimensional cancer invasion model which allows incorporation of tumor cells and stromal components to mimic in vivo conditions occurring in the bladder tumor microenvironment. This model provides the opportunity to observe the invasive process in real time using time-lapse imaging, interrogate the molecular pathways involved using confocal immunofluorescent imaging and screen compounds with the potential to block invasion. While this protocol focuses on bladder cancer, it is likely that similar methods could be used to examine invasion and motility in other tumor types as well.

The processes governing bladder cancer invasion represent opportunities for biomarker and therapeutic development. Here we present a bladder cancer invasion model which incorporates 3-D culture of tumor spheroids, time-lapse imaging and confocal microscopy. This technique is useful for defining the features of the invasive process and for screening therapeutic agents.

Bladder cancer is a significant health problem. It is estimated that more than 16,000 people will die this year in the United States from bladder cancer. ...

Generation of Tumor Organoids from Genetically Engineered Mouse Models of Prostate Cancer

Methods based on homologous recombination to modify genes have significantly furthered biological research. Genetically engineered mouse models (GEMMs) are a rigorous method for studying mammalian development and disease. Our laboratory has developed several GEMMs of prostate cancer (PCa) that lack expression of one or multiple tumor suppressor genes using the site-specific Cre-loxP recombinase system and a prostate-specific promoter. In this article, we describe our method for necropsy of these PCa GEMMs, primarily focusing on dissection of mouse prostate tumors. New methods developed over the last decade have facilitated the culture of epithelial-derived cells to model organ systems in vitro in three dimensions. We also detail a 3D cell culture method to generate tumor organoids from mouse PCa GEMMs. Pre-clinical cancer research has been dominated by 2D cell culture and cell line-derived or patient-derived xenograft models. These methods lack tumor microenvironment, a limitation of using these techniques in pre-clinical studies. GEMMs are more physiologically-relevant for understanding tumorigenesis and cancer progression. Tumor organoid culture is an in vitro model system that recapitulates tumor architecture and cell lineage characteristics. In addition, 3D cell culture methods allow for growth of normal cells for comparison to tumor cell cultures, rarely possible using 2D cell culture techniques. In combination, use of GEMMs and 3D cell culture in pre-clinical studies has the potential to improve our understanding of cancer biology.&#160;

We show a method for necropsy and dissection of mouse prostate cancer models, focusing on prostate tumor dissection. A step-by-step protocol for generation of mouse prostate tumor organoids is also presented.

Methods based on homologous recombination to modify genes have significantly furthered biological research. Genetically engineered mouse models (GEMMs) ...

Genetics

Prostate Organoid Cultures as Tools to Translate Genotypes and Mutational Profiles to Pharmacological Responses

Presented here is a protocol to study pharmacodynamics, stem cell potential, and cancer differentiation in prostate epithelial organoids. Prostate organoids are androgen responsive, three-dimensional (3D) cultures grown in a defined medium that resembles the prostatic epithelium. Prostate organoids can be established from wild-type and genetically engineered mouse models, benign human tissue, and advanced prostate cancer. Importantly, patient derived organoids closely resemble tumors in genetics and in vivo tumor biology. Moreover, organoids can be genetically manipulated using CRISPR/Cas9 and shRNA systems. These controlled genetics make the organoid culture attractive as a platform for rapidly testing the effects of genotypes and mutational profiles on pharmacological responses. However, experimental protocols must be specifically adapted to the 3D nature of organoid cultures to obtain reproducible results. Described here are detailed protocols for performing seeding assays to determine organoid formation capacity. Subsequently, this report shows how to perform drug treatments and analyze pharmacological response via viability measurements, protein isolation, and RNA isolation. Finally, the protocol describes how to prepare organoids for xenografting and subsequent in vivo growth assays using subcutaneous grafting. These protocols yield highly reproducible data and are widely applicable to 3D culture systems.

Presented here is a protocol to study pharmacological responses in prostate epithelial organoids. Organoids closely resemble in vivo biology and recapitulate patient genetics, making them attractive model systems. Prostate organoids can be established from wildtype prostates, genetically engineered mouse models, benign human tissue, and advanced prostate cancer.

Presented here is a protocol to study pharmacodynamics, stem cell potential, and cancer differentiation in prostate epithelial organoids. Prostate ...

Culture, Manipulation, and Orthotopic Transplantation of Mouse Bladder Tumor Organoids

The development of advanced tumor models has long been encouraged because current cancer models have shown limitations such as lack of three-dimensional (3D) tumor architecture and low relevance to human cancer. Researchers have recently developed a 3D in vitro cancer model referred to as tumor organoids that can mimic the characteristics of a native tumor in a culture dish. Here, experimental procedures are described in detail for the establishment of bladder tumor organoids from a carcinogen-induced murine bladder tumor, including culture, passage, and maintenance of the resulting 3D tumor organoids in vitro. In addition, protocols to manipulate the established bladder tumor organoid lines for genetic engineering using lentivirus-mediated transduction are described, including optimized conditions for the efficient introduction of new genetic elements into tumor organoids. Finally, the procedure for orthotopic transplantation of bladder tumor organoids into the wall of the murine bladder for further analysis is laid out. The methods described in this article can facilitate the establishment of an in vitro model for bladder cancer for the development of better therapeutic options.

This protocol provides detailed experimental steps to establish a three-dimensional in vitro culture of bladder tumor organoids derived from carcinogen-induced murine bladder cancer. Culture methods including passaging, genetic engineering, and orthotopic transplantation of tumor organoids are described.

The development of advanced tumor models has long been encouraged because current cancer models have shown limitations such as lack of three-dimensional ...

An Orthotopic Bladder Cancer Model for Gene Delivery Studies

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are needed. The tumor microenvironment can significantly influence tumor development and therapy response. It is therefore often desirable to grow tumor cells in the organ from which they originated. This protocol describes an orthotopic model of bladder cancer, in which MB49 murine bladder carcinoma cells are instilled into the bladder via catheterization. Successful tumor cell implantation in this model requires disruption of the protective glycosaminoglycan layer, which can be accomplished by physical or chemical means. In our protocol the bladder is treated with trypsin prior to cell instillation. Catheterization of the bladder can also be used to deliver therapeutics once the tumors are established. This protocol describes the delivery of an adenoviral construct that expresses a luciferase reporter gene. While our protocol has been optimized for short-term studies and focuses on gene delivery, the methodology of mouse bladder catheterization has broad applications.

Implantation of cancer cells into the organ of origin can serve as a useful preclinical model to evaluate novel therapies. MB49 bladder carcinoma cells can be grown within the bladder following intravesical instillation. This protocol demonstrates catheterization of the mouse bladder for the purpose of tumor implantation and adenoviral delivery.

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are ...

Medicine

Portal Vein Injection of Colorectal Cancer Organoids to Study the Liver Metastasis Stroma

Hepatic metastasis of colorectal cancer (CRC) is a leading cause of cancer-related death. Cancer-associated fibroblasts (CAFs), a major component of the tumor microenvironment, play a crucial role in metastatic CRC progression and predict poor patient prognosis. However, there is a lack of satisfactory mouse models to study the crosstalk between metastatic cancer cells and CAFs. Here, we present a method to investigate how liver metastasis progression is regulated by the metastatic niche and possibly could be restrained by stroma-directed therapy. Portal vein injection of CRC organoids generated a desmoplastic reaction, which faithfully recapitulated the fibroblast-rich histology of human CRC liver metastases. This model was tissue-specific with a higher tumor burden in the liver when compared to an intra-splenic injection model, simplifying mouse survival analyses. By injecting luciferase-expressing tumor organoids, tumor growth kinetics could be monitored by in vivo imaging. Moreover, this preclinical model provides a useful platform to assess the efficacy of therapeutics targeting the tumor mesenchyme. We describe methods to examine whether adeno-associated virus-mediated delivery of a tumor-inhibiting stromal gene to hepatocytes could remodel the tumor microenvironment and improve mouse survival. This approach enables the development and assessment of novel therapeutic strategies to inhibit hepatic metastasis of CRC.

Portal vein injection of colorectal cancer (CRC) organoids generates stroma-rich liver metastasis. This mouse model of CRC hepatic metastasis represents a useful tool to study tumor-stroma interactions and develop novel stroma-directed therapeutics such as adeno-associated virus-mediated gene therapies.

Hepatic metastasis of colorectal cancer (CRC) is a leading cause of cancer-related death. Cancer-associated fibroblasts (CAFs), a major component of the ...

Ex Vivo Organoid Model of Adenovirus-Cre Mediated Gene Deletions in Mouse Urothelial Cells

Bladder cancer is an understudied area, particularly in genetically engineered mouse models (GEMMs). Inbred GEMMs with tissue-specific Cre and loxP sites have been the gold standards for conditional or inducible gene targeting. To provide faster and more efficient experimental models, an ex vivo organoid culture system is developed using adenovirus Cre and normal urothelial cells carrying multiple loxP alleles of the tumor suppressors Trp53, Pten, and Rb1. Normal urothelial cells are enzymatically disassociated from four bladders of triple floxed mice (Trp53f/f: Ptenf/f: Rb1f/f). The urothelial cells are transduced ex vivo with adenovirus-Cre driven by a CMV promoter (Ad5CMVCre). The transduced bladder organoids are cultured, propagated, and characterized in vitro and in vivo. PCR is used to confirm gene deletions in Trp53, Pten, and Rb1. Immunofluorescence (IF) staining of organoids demonstrates positive expression of urothelial lineage markers (CK5 and p63). The organoids are injected subcutaneously into host mice for tumor expansion and serial passages. The immunohistochemistry (IHC) of xenografts exhibits positive expression of CK7, CK5, and p63 and negative expression of CK8 and Uroplakin 3. In summary, adenovirus-mediated gene deletion from mouse urothelial cells engineered with loxP sites is an efficient method to rapidly test the tumorigenic potential of defined genetic alterations.

Ex Vivo Organoid Model of Adenovirus-Cre Mediated Gene Deletions in Mouse Urothelial Cells

This protocol describes the process of the generation and characterization of mouse urothelial organoids harboring deletions in genes of interest. The methods include harvesting mouse urothelial cells, ex vivo transduction with adenovirus driving Cre expression with a CMV promoter, and in vitro as well as in vivo characterization.

Bladder cancer is an understudied area, particularly in genetically engineered mouse models (GEMMs). Inbred GEMMs with tissue-specific Cre and loxP sites ...

Generation and Culturing of High-Grade Serous Ovarian Cancer Patient-Derived Organoids

Organoids are 3D dynamic tumor models that can be grown successfully from patient-derived ovarian tumor tissue, ascites, or pleural fluid and aid in the discovery of novel therapeutics and predictive biomarkers for ovarian cancer. These models recapitulate clonal heterogeneity, the tumor microenvironment, and cell-cell and cell-matrix interactions. Additionally, they have been shown to match the primary tumor morphologically, cytologically, immunohistochemically, and genetically. Thus, organoids facilitate research on tumor cells and the tumor microenvironment and are superior to cell lines. The present protocol describes distinct methods to generate patient-derived ovarian cancer organoids from patient tumors, ascites, and pleural fluid samples with a higher than 97% success rate. The patient samples are separated into cellular suspensions by both mechanical and enzymatic digestion. The cells are then plated utilizing a basement membrane extract (BME) and are supported with optimized growth media containing supplements specific to the culturing of high-grade serous ovarian cancer (HGSOC). After forming initial organoids, the PDOs can sustain long-term culture, including passaging for expansion for subsequent experiments.

Patient-derived organoids (PDO) are a three-dimensional (3D) culture that can mimic the tumor environment in vitro. In high-grade serous ovarian cancer, PDOs represent a model to study novel biomarkers and therapeutics.

Organoids are 3D dynamic tumor models that can be grown successfully from patient-derived ovarian tumor tissue, ascites, or pleural fluid and aid in the ...

Establishment and Culture of Patient-Derived Breast Organoids

Breast cancer is a complex disease that has been classified into several different histological and molecular subtypes. Patient-derived breast tumor organoids developed in our laboratory consist of a mix of multiple tumor-derived cell populations, and thus represent a better approximation of tumor cell diversity and milieu than the established 2D cancer cell lines. Organoids serve as an ideal in vitro model, allowing for cell-extracellular matrix interactions, known to play an important role in cell-cell interactions and cancer progression. Patient-derived organoids also have advantages over mouse models as they are of human origin. Furthermore, they have been shown to recapitulate the genomic, transcriptomic as well as metabolic heterogeneity of patient tumors; thus, they are capable of representing tumor complexity as well as patient diversity. As a result, they are poised to provide more accurate insights into target discovery and validation and drug sensitivity assays. In this protocol, we provide a detailed demonstration of how patient-derived breast organoids are established from resected breast tumors (cancer organoids) or reductive mammoplasty-derived breast tissue (normal organoids). This is followed by a comprehensive account of 3D organoid culture, expansion, passaging, freezing, as well as thawing of patient-derived breast organoid cultures.

A detailed protocol is provided here for establishing human breast organoids from patient-derived breast tumor resections or normal breast tissue. The protocol provides comprehensive step-by-step instructions for culturing, freezing, and thawing human patient-derived breast organoids.

Breast cancer is a complex disease that has been classified into several different histological and molecular subtypes. Patient-derived breast tumor ...

Culture of Bladder Cancer Organoids as Precision Medicine Tools

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